BioLegend Western Blot Troubleshooting Guide |
What's the issue? |
Possible Causes |
Solution |
No or low Signal |
The primary and secondary antibodies are not compatible. |
Ensure you are using secondary antibody that binds to your primary antibody (i.e. if your primary is rat, be sure you are using an anti rat secondary). |
Insufficient primary or secondary antibody has bound to the protein of interest. |
Utilize a higher concentration of antibody and or incubate for a longer period (i.e. overnight at 4°C). |
There is not enough antigen. |
Load a larger amount of protein onto the gel. Use protease inhibitors and run the recommended positive control. |
Overuse of primary antibody. |
Use fresh antibody (the effective concentration is lowered after each use). |
Incubation with detection reagent not sufficient. |
Increase the blots incubation time with detection reagent. |
Detection reagents are not working. |
Make sure detection reagents are functional by testing with a different primary antibody. |
Poor transfer during blotting. |
Make sure the transfer apparatus is set up correctly. Ensure you are using the correct transfer times. |
Secondary antibody is inhibited by sodium azide. |
Do not use sodium azide with HRP-Conjugated antibodies. |
Excessive membrane washing. |
Reduce washing step repetitions or duration. |
Target protein ran off the gel. |
Use a positive control and a molecular weight marker matched to the size range of the target protein. |
The target protein is not found in high concentrations in your sample. |
Maximize the target's concentration by enriching the sample beforehand. |
The primary or secondary antibody binds to the blocking agent. |
Utilize a mild detergent or switch to a different blocking reagent. |
Poor binding of proteins to membrane. |
Use a membrane with the correct binding capacity. Dry PVDF membranes after transfer to promote strong binding. |
The protein in the species tested is not recognized by your primary antibody. |
Run a positive control. Check literature or perform a BLAST alignment to see whether your antibody should react with the target protein. |
High Background |
Insufficient washing or blocking. |
Increase blocking time or consider using an alternate blocking reagent. Increase the number of washes. |
Concentration of the primary antibody is too high. |
Determine optimal antibody concentration through titration. Use a more dilute antibody with longer incubation times (slow targeted binding is best). |
Secondary antibody binding non-specifically, or binding with blocking agent. |
Run a secondary control with no primary antibody. |
Overloaded protein. |
Decrease the amount of protein loaded on the gel, or dilute the sample. |
Contamination of equipment or reagents. |
Replace reagents, ensure all equipment is properly cleaned. |
Membrane causing high background. |
PVDF membranes are considered to give higher background than nitrocellulose membranes. |
Membrane dried out during incubation. |
Ensure membrane is not drying out during the incubation period. |
Incubation temp too high. |
Incubate membrane at 4°C. |
Cross-reactivity of phospho-specific antibodies with blocking agent. |
The user may have to try different blocking buffers, such as milk, BSA, etc. to reduce non-specific binding while maintaining specific signals. For optimal results, follow the blocking buffer recommendations from your antibody provider. |
Multiple or non-specific bands |
Primary antibody concentration too high. |
Decrease the concentration of primary antibody. Run secondary control without the primary antibody. |
Excess protein on gel. |
Reduce the amount of protein loaded. |
Issues with blocking. |
Optimize blocking time and blocking reagent. |
Insufficient washing. |
Increase number of wash steps. |
Antibody not properly purified. |
Use antibodies purified by the affinity method. |
Target protein has been degraded. |
Use fresh sample. Include protease inhibitors in your sample buffer. |
Frequently passaged cell lines accumulate differences in protein expression profiles. |
Retrieve and expand original cell line, run samples in parallel. |
Target protein has several modified forms (acetylation, methylation, glycosylation etc.). |
Refer to literature, use agent to remove modifications when possible/necessary. |
Protein subtypes have different molecular weights. |
Use bioinformatics analysis and review literature to estimate the correct protein size. |
There are splice variants from the same protein family that share similar epitopes. |
Check literature and/or perform a blast search to confirm. |
Multimer formation of target protein. |
Prior to SDS page, boil protein for 10 min to disrupt multimers. |
Diffuse Bands |
Concentration of antibody too high. |
Reduce antibody concentration. |
Protein transfer too rapid or gel became over-heated during electrophoresis. |
Increase the transfer time and/or run gel at 4°C |
Too much protein loaded on gel. |
Decrease the quantity of protein loaded on gel. |
Smile effect on bands |
Migration was too rapid. |
Decrease voltage when running gel. |
Temperature during migration was too high. |
Run gel at 4°C. |
Uneven staining of gel |
Bacterial contamination of antibodies. |
Store antibodies as recommended by your antibody provider. Use fresh buffers. |
Insufficient antibody volume. |
Ensure that the membrane is completely covered with antibody and incubate with agitation. |
Target band is extremely high/low on blot |
Separation during electrophoresis not efficient. |
Change gel percentage: use a lower percentage for large proteins, a higher percentage for small proteins. |
Lane with protein ladder is black |
The antibody is reacting with the protein ladder. |
Load gel so there is a blank lane between the ladder and the first sample lane. |
Uneven white spots on blot |
Air bubbles were trapped between the gel and membrane during transfer. |
Ensure air bubbles are removed when preparing for transfer. |
Black dots on the blot |
Antibodies are binding to blocking agent. |
Filter blocking agent. |
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